Peptides and medical uses thereof

ABSTRACT

The present invention relates to peptides, a composition comprising said peptides and the use thereof as inhibitors of angiogenesis and/or neoangiogenesis. Furthermore, the present invention relates to the use of said peptides and said composition for the treatment of pathologies correlated with an incorrect angiogenesis and/or neoangiogenesis. In particular, in this context reference is made to angiogenesis and/or neoangiogenesis correlated with VEGFR1.

TECHNICAL FIELD

The present invention relates to peptides, a composition comprising saidpeptides and the use thereof as inhibitors of angiogenesis and/orneoangiogenesis. Furthermore, the present invention relates to the useof said peptides and said composition for the treatment of pathologiescorrelated with an incorrect angiogenesis and/or neoangiogenesis. Inparticular, in this context reference is made to angiogenesis and/orneoangiogenesis correlated with VEGFR1.

PRIOR ART

Given the considerable severity and wide spectrum of pathologies forwhich inhibition of the activation of VEGFR-1 may have application, itis conceivable that there will be a strong demand for syntheticcompounds capable of binding VEGFR-1 and able to interfere in theinteraction between the VEGF-A, PIGF, VEGF-B ligands and VEGF-A/PIGFheterodimer with VEGFR-1. In fact, advantageously, synthetic compoundsare intrinsically free of contaminants of biological origin and they canalso be produced at a considerably lower cost than biotherapeutics ofrecombinant origin.

With the aim of neutralizing ligands, many therapeutic approaches usemonoclonal antibodies because they are molecules characterized by highspecificity and affinity. However, synthetic molecules, too, have theiradvantages, because they are easier and more inexpensive to produce,more stable and more easily deliverable.

In this regard, Ponticelli et al. recently described, in 2008, atetrameric tripeptide selected from a peptide library, in which apeptide chain with the formula (R-Glu)-(S-Cys(Bzl))-(S-Cha) wastetramerized on a “core” of three lysines (Tam, J. P. 1988. Proc. Natl.Acad. Sci. USA 85:5409-5413).

The tetrameric peptide has the following structure:

The scientific evidence reported by Ponticelli et al. demonstrates thatthe above-mentioned tetrameric peptide is capable of binding VEGFR1 andinhibiting, in vitro, the interaction of PIGF, VEGF-A and VEGF-B with anIC50 of about 10 μM. Furthermore, the peptide is not capable of bindingVEGFR-2 and does not interfere in its activation by VEGF-A.

Finally, the peptide:

1) has shown anti-angiogenic activity in vitro, interfering with thepro-angiogenic activity of PIGF and VEGF-A;2) is able to displace the VEGF-A-sFlt1 bond in thecornea—non-vascularized under physiological conditions—consequentlyrendering it VEGF-A free and capable of promoting neoangiogenesis;3) when administered intraperitoneally, reduces tumor growth,angiogenesis and arteriogenesis as well as metastatization; and4) when administered intravitreally, reduces choroidalneovascularization (Cicatiello et al. 2015).

The anti-angiogenic activity of the peptide is due both to an inhibitionof the formation of new blood vessels and the capacity to inhibit therecruitment of inflammatory cells, preferably monocytes-macrophages, atthe sites of neoangiogenesis.

The anti-arteriogenic activity is based on the capacity to inhibit therecruitment of smooth muscle cells at the sites of neoangiogenesis.

OBJECT OF THE INVENTION

In this context, the authors of the present invention have surprisinglyfound that by inserting, at the C-Terminal of the peptide, a chemicalgroup, in particular an amino acid characterized by a side chain havinga steric hindrance comparable to that of the thiol or thioether group,one significantly improves the activity of the molecule.

In fact, the above-mentioned modifications do not compromise selectivebinding with VEGFR1 and the capacity to compete, in a dose-dependentmanner, with VEGF-A and/or PIGF in binding with VEGFR1. On the contrary,these modifications are capable of producing a 50% inhibition (IC50) ofthe interaction between PIGF or VEGF and VEGFR1 at a concentration ofless than 1000 nM; this is a wholly unexpected result considering thatthe affinity of the peptide reported in Ponticelli et al. towards VEGFR1is equal to 10000 nM or higher (expressed as IC50). In other words, thepeptides of the present invention have an inhibitory capacity which isabout one order of magnitude greater than that of the peptide reportedin Ponticelli et al.

Furthermore, the authors of the present invention have surprisinglyfound that, when administered orally, or by gavage, both the peptidedescribed in Ponticelli et al. and the peptides of the present inventionhave demonstrated a significant capacity to inhibit choroidalneovascularization. Therefore, these molecules are therapeuticallyeffective for treating, preferably by oral administration, pathologiescorrelated with or in any case caused by an alteration of angiogenesis,preferably VEGFR1-dependent angiogenesis.

A detailed description of the invention follows, along with non-limitingillustrative examples which make reference to the figures anddefinitions below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibitory activity of iVR1 and iVR1-Cys and of theanti-PIGF monoclonal antibody with reference to PIGF-inducedphosphorylation of VEGFR-1. The analysis of VEGFR-1 phosphorylationinduced with 20 ng/ml of PIGF was conducted on 293-VEGFR-1 cells bywestern blotting. iVR1-Cys and iVR1 were added simultaneously to PIGF ata concentration of 5 μM. A human anti-PIGF neutralizing monoclonalantibody was used at a concentration of 3.3 nM as an inhibition control.PBS was used as a negative control.

FIG. 2 shows that intravitreally administered iVR1-Cys inhibitslaser-induced choroidal neovascularization in a dose-dependent manner. Asingle intravitreal injection of 10 or 50 μg of iVR1-Cys brings about adose-dependent reduction of choroidal neovascularization equal to 48.9%and 75.9% compared to injection of the vehicle (DMSO). The same amountsof iVR1 bring about an inhibition of CNV equal to 37.8% and 73.9%. Thecontrol peptide (PC) shows no inhibitory capacity. Quantization of thevolume of neovascularization was performed on n=12 and 15 spots for iVR110 μg and 50 μg; on n=10 and 8 spots for iVR1-Cys 10 μg and 50 μg; n=15spots for PC and n=14 spots for the DMSO. The data are represented asthe mean±SEM relative to the control. #p<0.05; * p>0.0002; ¶p<0.02; §p>0.002; vs PC and DMSO. At the bottom, images representative of CNV.The bar represents 100 μm.

FIG. 3 shows that orally administered iVR1-Cys inhibits laser-inducedchoroidal neovascularization. Oral administration of iVR1-Cys at 50mg/Kg twice a day for seven days brings about a 45.9% reduction ofchoroidal neovascularization, compared to the vehicle. The same amountof iVR1 brings about a similar inhibition of CNV (49.7%). Quantizationof the volume of neovascularization was performed on n=18 spots foriVR1-Cys, n=20 spots for iVR1, and n=10 spots for the vehicle. The dataare represented as the mean±SEM relative to the control. *p=0.001 and §p=0.007 vs DMSO. At the bottom, images representative of CNV. The barrepresents 100 μm.

DEFINITIONS

In this context, the term “VEGF” means vascular endothelial growthfactor. In humans there exist 5 different vascular endothelial growthfactors, VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF, encoded by fivedifferent genes. All are glycosylated dimeric proteins.

In this context, the term “VEGF-A” means vascular endothelial growthfactor-A, formerly also known as VPF (vascular permeability factor). Itis the most potent factor of the VEGF family, with a decisive role inboth physiological and pathological angiogenesis. At least six differentisoforms obtained by alternative splicing have been described in humans.All are capable of interacting with two receptors, which are calledVEGFR-1 and VEGFR-2.

In this context, the term “PIGF” means placental growth factor, whoserole is confined to the conditions of angiogenesis associated withpathological states. Four different isoforms have been described inhumans. All are capable of specifically binding VEGFR-1. VEGF-A and PIGFact in strong synergism in pathological conditions, because bothinteract with VEGFR-1 and because when the two respective genes areexpressed in the same cell, they are able to give rise to VEGF-A/PIGFheterodimers capable of interacting with VEGFR-1 or inducingVEGFR-1/VEGFR-2 heterodimerization.

In this context, the term “VEGFR-1” means VEGF receptor 1, also known asFlt-1. VEGFR-1 has an intracellular tyrosine-kinase domain, whilst theextracellular portion consists of seven IgG-like domains. VEGF-A,VEGF-B, or PIGF bring about dimerization of the receptor with aconsequent activation by autophosphorylation of the tyrosine-kinasedomains. Besides being expressed in endothelial cells, VEGFR-1 isexpressed in many other types of cells, including smooth muscle cells,monocytes-macrophages, fibroblasts and endothelial precursors. It has afundamental role in recruiting the different types of cells thatcontribute to angiogenesis. In this context, the term “soluble VEGFR-1”(sVEGFR-1) means the soluble form of VEGF receptor 1, also known assFlt-1. It consists of the first six IgG-like extracellular domains ofVEGFR-1 plus a tail and is generated from the VEGFR-1 gene byalternative splicing. It is normally expressed by the same cells inwhich the full-length form of VEGFR-1 is expressed, with the exceptionof the cornea, in which the soluble form is preferentially expressed,being decisive for maintaining the cornea in an avascular state. Themessenger sequences of full-length and soluble human VEGFR1 arepreferably SEQ ID NO: 1 and 2, respectively, whereas the proteinsequences of full-length human VEGFR1 are SEQ ID NO: 3 and 4,respectively. Sequences characterized by an identity to the sequencesdescribed herein ranging from 80-99.9% must be considered part of thepresent description.

In this context, the term “VEGFR-2” means VEGF receptor 2, also known asKDR in humans and Flk-1 in mice. VEGFR-2 is specifically bound byVEGF-A, and has an organization in domains and an activation mechanismsimilar to the ones described for VEGFR-1. Unlike receptor 1, it isessentially expressed in endothelial cells. It has a fundamental role instimulating the proliferation, migration and differentiation ofendothelial cells.

In this context, the term “angiogenesis” means the process of formationof new blood vessels from pre-existing vessels; in this contextangiogenesis is preferably referred to as a process of formation of newblood vessels associated with pathological conditions of various types,preferably selected from:

-   -   neovascular eye diseases, preferably selected from: macular        edema, the wet form of age-related macular degeneration,        diabetic retinopathy, retinopathy of prematurity, retinopathy of        central retinal vein occlusion, vitreous hemorrhage and retinal        detachment and combinations thereof; and/or;    -   solid tumors and/or tumor metastatization, said tumors        preferably being selected from: leukemia and lymphomas,        preferably acute lymphocytic leukemia, acute non-lymphocytic        leukemia, chronic lymphocytic leukemia, multiple myeloma,        Hodgkin's lymphoma, Hodgkin's disease, infantile or adult solid        tumors, brain tumors, neuroblastoma, retinoblastoma, Wilms        tumor, osteosarcomas and chondrosarcomas, lung tumors,        colorectal cancer, breast cancer, prostate cancer, uterine        cancer, ovarian cancer, urinary system cancer, bladder cancer,        tumor of the oral cavity, tumor of the pancreas, melanoma and        tumors of the skin, tumor of the stomach, tumor of the brain,        tumor of the thyroid, tumor of the larynx, tumor of the liver,        tumor of the testicles; and/or    -   diseases of the bones or joints, preferably selected from:        rheumatoid arthritis, synovitis, cartilage and/or bone        destruction, osteomyelitis, hypertrophy and/or hyperplasia of        the synovial tissue, formation of osteophytes, neoplasms and/or        metastases and combinations thereof; and/or    -   pathologies of blood vessels, preferably selected from:        atherosclerosis, hemangioma, hemangioendothelioma and        combinations thereof; and/or    -   skin diseases, preferably selected from: psoriasis, warts,        pyogenic granulomas, hair growth, Kaposi's sarcoma, keloids of        wounds, allergic edema, neoplasms and combinations thereof;        and/or    -   angiogenesis observed in pathologies of adipose tissue,        preferably obesity; and/or    -   diabetes and/or its consequences, preferably retinopathy and/or        diabetic foot; and/or    -   diseases of hematopoiesis, preferably AIDS and/or Kaposi's        sarcoma.        In this context, the term “neoangiogenesis” means new        angiogenesis, preferably with reference to the formation of new        blood vessels in tissues in which they were previously absent        and/or an increase in the number of blood vessels in already        vascularized tissues; in this context, the neoangiogenesis is        preferably dependent on the activity of VEGFR-1.

In this context, the term “vascularization” means angiogenesis, i.e.,they are used as synonyms.

In this context, the term “neovascularization” means neoangiogenesis,preferably dependent on the activity of VEGFR-1.

In this context, the term “arteriogenesis” means the process ofstabilization of new blood vessels through the covering of the vesselswith smooth muscle cells.

In this context, “inhibitor” means a chemical and/or biological entitycapable of antagonizing the activity of a receptor by binding thereceptor itself and/or the soluble ligands thereof, thus preventingtheir interaction.

In this context, the term “effective dose” means a dosage intervalwithin which the administration of the active substance described in theinvention is capable of determining the desired biological effect. As iswell known to the person skilled in the art, it may vary depending on:state of health, physical condition of the individual who needs to betreated, age, the formulation of the active substance, the assessment ofthe physician taking care of the patient, the ability of the system ofthe single individual to respond effectively, degree of responsedesired, taxonomic group (for example, human, non-human primate,primate, etc.), and other relevant factors. It is expected that theeffective dose of the active substance described in the invention willfall within an interval that is sufficiently wide to be determined withroutine tests. Generally, as reported by Ragan-Shaw et al. (FASEB J.2008 Mar.; 22(3):659-61), and thus in this context as well, theeffective dose administered preferably ranges between 10 and 2000mg/dose when administered preferably systemically, preferablysystemically by the enteral route, more preferably orally, sublinguallyor rectally. Alternatively, the effective dose administered rangesbetween 1 and 100 mg/dose when administered preferably intravitreally.Alternatively, the effective dose administered preferably ranges between0.16 and 33.3 mg/kg of body weight. The treatment program provides for asingle dose or multiple doses.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

A first aspect of the invention refers to peptides, preferablymultimeric peptides, isolated and characterized by the following generalformula (II):

{{{[Y1-Glu-Cys(Bzl)-Cha]2-Z1}i-Z2}j-Z3}z-Y2-Y3   (Formula II)

wherein

-   -   Y1 is the amino-terminal function of the peptide (NH₂) or at        least one chemical group preferably selected in Table I. The        list is understood also to include chemical groups, preferably        amino acids, which possess a steric hindrance and/or chemical        properties, in particular a side chain in the case of amino        acids, which mimic those of the chemical groups, preferably the        amino acids, listed in Table I and/or which are characterized by        a similarity, preferably of at least 70%, said similarity being        determined with methods known to the person skilled in the art,        for example, but not exclusively, with the methods described in        Woong-Hee Shin et al., Molecules 2015, 20, 12841-12862.

In this context, it should be clarified that the D/L notations suitablefor defining the absolute configuration of the chiral centers present inthe groups of the present description are interchangeable with the R/Snotation following rules reported in the literature, as is known to theperson skilled in the art.

TABLE I N. 3-letter code Abbreviation  1 D-Alanine D-Ala  2 D-AsparticAcid D-Asp  3 D-Valine D-Val  4 D-Glutamic Acid D-Glu  5L-Cyclohexylalanine L-Cha  6 D-Phenylalanine D-Phe  7 D-Threonine D-Thr 8 D-Methionine D-Met  9 D-Lysine D-Lys 10 D-Cysteine(S- D-Cys(Acm)acetamidomethyl) 11 D-Tyrosine D-Tyr 12 D-Proline D-Pro 13 D-LeucineD-Leu 14 D-Arginine D-Arg 15 D-Asparagine D-Asn 16 D-Isoleucine D-Ile 17D-Arginine(N^(┌)-Tosyl) D-Arg(Tos) 18 D-Serine D-Ser 19L-Cysteine(S-benzyl) L-Cys(Bzl) 20 L-Cysteine(S- L-Cys(Acm)acetamidomethyl) 21 D-Histidine D-His 22 D-Glutamine D-Gln 23D-Tryptophan D-Trp 24 L-Glutamic-(β-allyl) Acid L-Glu(β-OAll) 25β-Alanine β-Ala 26 L-Cysteine(S-p- L-Cys(p-MeBzl) methyl-benzyl) 27L-Cysteine(S-tert-butyl) L-Cys(tBu) 28 L-Methionine-sulfone L-Met(O)2 29L-Methionine-sulfoxide L-Met(O) 30 Glycine Gly

-   -   Glu indicates glutamic acid, preferably in an absolute        configuration R on the Cα of the amino acid (R-Glu).    -   Cys(Bzl) indicates benzyl cysteine, preferably in an absolute        configuration S on the Cα of the amino acid containing a        sulfur-linked benzyl group of the amino acid        (S-benzyl-cysteine/S-Cys (Bzl) side chain.    -   Cha indicates cyclohexylalanine, preferably in an absolute        configuration S on the Cα of the amino acid        (S-cyclohexylalanine/S-Cha).    -   Y2 is preferably selected from:        -   1. the tripeptide R-Glu-S-Cys(Bzl)-S-Cha, and        -   an α-amino acid, preferably selected from a glycine or an            α-amino acid characterized by at least one thiol or            thioether group, said α-amino acid characterized by at least            one thiol or thioether group preferably being selected from            the ones shown in Table II and combinations thereof.

The list is understood also to include chemical groups, preferably aminoacids, which possess a steric hindrance and/or chemical properties, inparticular a side chain in the case of amino acids, which mimic those ofthe chemical groups, preferably the amino acids, listed in Table II andwhich are characterized by a similarity, preferably of at least 70%,said similarity being determined with methods known to the personskilled in the art, for example, but not exclusively, with the methodsdescribed in Woong-Hee Shin et al., Molecules 2015, 20, 12841-12862.

TABLE II N. Y  1 D-cysteine  2 L-cysteine  3 L-homocysteine  4D-homocysteine  5 D-cysteine-S-methyl  6 L-cysteine-S-methyl  7D-cysteine-S-ethyl  8 L-cysteine-S-ethyl  9 L-methionine 10 D-methionine11 D-cysteine-S-benzyl 12 L-cysteine-S-benzyl 13 L-methionine sulfoxide14 D-methionine sulfoxide 15 L-methionine sulfone 16 D-methioninesulfone 17 D-serine 18 L-serine 19 D-serine-O-methyl 20L-serine-O-methyl 21 D-serine-O-ethyl 22 L-serine-O-ethyl 23D-serine-O-benzyl 24 L-serine-O-benzyl 25 D-threonine 26 L-threonine 27D-threonine-O-methyl 28 L-threonine-O-methyl 29 D threonine-O-ethyl 30L-threonine-O-ethyl 31 D-threonine-O-benzyl 32 L-threonine-O-benzyl

-   -   Y3 is preferably selected from: a carboxylic group, a        carboxyamide group, an N-methyl-substituted carboxyamide or        di-substituted N, N-dimethyl group, a hydroxyl group and a        hydrogen.    -   Z1, Z2 and Z3 preferably indicate a trifunctional group,        preferably characterized by the following formula (III):

where k is an integer, preferably comprised between 1 and 4, and B ispreferably an amino group or a hydroxyl group. Said trifunctionalmolecule is preferably in an R or S absolute configuration.

Preferably, Z1, Z2 and Z3 are used for the purpose of obtaining abranched structure. In fact, this type of structure is generally used tomultimerize peptides following known methods for this purpose, forexample when B is an amino group, the method described by Tam et al. canbe used (Tam J. P., 1988, PNAS, 85, 5409-5413).

Z1, Z2 and Z3 can be assembled in such a way as to obtain a structure offormula (II) with multiple groups Z1, Z2 and Z3, preferably containing1, 3 or 7 trifunctional molecules.

According to a preferred embodiment of the invention, Z1 and/or Z2and/or Z3 are joined to one another preferably by amide bonds in such away as to form a branched structure. Alternatively, they can be joinedto one another by an ester bond, for example when B is preferably ahydroxyl group.

-   -   i is preferably 4, 2 or 1.    -   j is preferably 2, 1 or 0.    -   z is preferably 1 or 0.

According to a preferred embodiment, when i=4, j=2 and z=1. According toa further preferred embodiment, when i=2, j=1 and z=0.

According to a further preferred embodiment, when i=1, j=z=0.

If j=0 the Z2 group is omitted and if z=0 the Z3 group is omitted.

For the purposes of the present invention, the particularly preferredembodiment envisages that i is equal to 2, j is equal to 1 and Z2 is 0or omitted (in other words, Z3 is not present, i.e., it is absent).

In the particularly preferred embodiment of the invention, Z1, Z2 and Z3are a R- or S-lysine (k=4) and i is preferably equal to 2.

The preferred formula of the multimeric peptide of the invention isrepresented by the formula below (Figure IIa):

According to a particularly preferred embodiment of the invention, thepeptide is a tetrameric peptide characterized by the formula (IIb):

In which:

-   -   Y1 is a hydrogen atom;    -   Y2 is a D-cysteine;    -   Y3 is an unsubstituted primary amide group    -   Z1, Z2 and Z3 being as defined above;    -   i equal to 2;    -   j equal to 1; and    -   z equal to zero, i.e., absent.

For the sake of convenience, the particularly preferred embodiment ofthe peptide characterized by the formula IIb will be called iVR1-Cysfrom this moment on.

The above-described peptides show a biological activity, preferably amodulation activity, more preferably an activity of inhibitingangiogenesis and/or neoangiogenesis, which is improved compared to thatof the peptide described by Ponticelli et al. as reported and discussedbelow in the experimental results which—in this context—have anon-limiting illustrative purpose. The angiogenesis and/orneoangiogenesis being referred to in this context is preferablyVEGFR1-dependent as earlier defined.

The peptide described in Ponticelli et al. is also a tetrameric peptidecharacterized by the formula (IIc):

Wherein:

-   -   Y1 is a hydrogen atom;    -   Y2 is a glycine;    -   Y3 is an unsubstituted primary amide group    -   Z1, Z2 and Z3 being as defined above;    -   i equal to 2;    -   j equal to 1; and    -   z equal to zero.

For the sake of convenience, the particularly preferred embodiment ofthe peptide characterized by the formula IIc will be called iVR1 fromthis moment on.

The authors of the present invention have surprisingly found that bymodifying IVR1, in particular at the terminal carboxyl, preferably byinserting an R-Glu-S-Cys(Bzl)-S-Cha group or an α-amino acid, preferablyselected from an α-amino acid characterized by at least one thiol orthioether group, said α-amino acid characterized by at least one thiolor thioether group preferably being selected from the ones shown inTable II and combinations thereof, one obtains peptides characterized byan improved biological activity, preferably an improved modulationcapacity, preferably by inhibiting angiogenesis and/or neoangiogenesisas defined above.

In fact, as shown and discussed in greater detail in the examples,iVR1-Cys has demonstrated a capacity to inhibit, in a dose-dependentmanner, the interaction of both PIGF and VEGF-A with VEGFR-1, a capacitywhich is improved compared to iVR1. In particular, the concentration atwhich iVR1-Cys is capable of inhibiting the interaction of PIGF withVEGFR-1 by 50% (IC50) is below 1000 nM, whereas the IC50 forVEGF-A/VEGFR-1 inhibition is close to or just above 1000 nM. iVR1, onthe other hand, is capable of inhibiting the interaction of PIGF withVEGFR-1 by 50% (IC50) at a concentration close to 10000 nM. Similarly,the IC50 for VEGF-A/VEGFR-1 inhibition by iVR1 is close to or just above10000 nM.

Therefore, iVR1-Cys shows an inhibitory capacity that is 10 timesgreater than the one reported for iVR1.

Furthermore, the authors have demonstrated—with in vivo assays—that iVR1brings about a 37.8% and 39.3% inhibition of choroidalneovascularization vs the vehicle and PC (p<0.05), whereas iVR1-Cysbrings about a 48.9% and 51.0% inhibition vs the vehicle and PC(p<0.02). Therefore, iVR1-Cys shows a greater inhibition effectivenessthan the peptide iVR1, as it brings about a further 19.3% reduction ofneovascularization.

Finally, when administered orally, or by gavage, both peptides tested byway of example are capable of inducing a significant inhibition ofneovascularization compared to the vehicle.

The latter fact is particularly relevant because although Ponticelli etal. and Cicatiello et al. 2015 had already demonstrated the capacity ofiVR1 to inhibit choroidal angiogenesis and neovascularization byintravitreal injection, it was absolutely not expected thatadministering the peptides through different routes, in particular bygavage, could maintain or even improve the therapeutic effectiveness,above all in the case of a highly complex organ like the eye andpathologies affecting it caused by or in any case correlated with anunregulated, preferably increased angiogenesis/neoangiogenesis. Inparticular, the neovascular diseases of the eye to which reference isbeing made are preferably selected from: macular edema, the wet form ofage-related macular degeneration, diabetic retinopathy, retinopathy ofprematurity, central retinal vein occlusion, vitreous hemorrhage andretinal detachment and combinations thereof.

In the light of this evidence, it is clear that the administration ofthe peptides of the invention through the oral route, or by gavage, istherapeutically effective also for treating pathologies, such as cancerfor example, which are in general correlated withangiogenesis/neoangiogenesis. The angiogenesis or neoangiogenesis towhich reference is being made is preferably VEGFR1-dependent.

According to one embodiment of the invention, the peptides can bemodified in order to facilitate or improve delivery, preferably byPEGylation, or using container/shuttle/carrier systems, preferablyliposomes, micelles, capsules, emulsions, matrices, gels and the like.

A further aspect of the present invention relates to a compositioncomprising the peptides as described in detail and at least one furtherpharmaceutically accepted ingredient.

The composition preferably comprises at least one peptide characterizedby Formula IIa, more preferably the peptide characterized by FormulaIIb, i.e., iVR1-Cys.

In this context, pharmaceutically accepted ingredient means a compoundselected from: excipients, diluents, carriers, adjuvants, preservatives,antibiotics, anti-inflammatories, oils, vitamins, antioxidants,chelating agents, solubilizing agents, viscosity agents, inert gases,surfactant agents, emulsifying agents, buffer substances,immunosuppressants, anti-tumor agents and combinations thereof.

For example, according to one embodiment, the composition comprises thepeptides of the invention in combination with: at least oneanti-angiogenic/anti-neoangiogenic molecule, an antibody neutralizingthe action of PIGF, at least one anti-VEGFR-1, anti-VEGFR-2,anti-VEGFR-3 antibody, at least one anti-VEGF-A, anti-VEGF-B,anti-VEGF-C, anti-VEGF-D, anti-VEGF-E antibody and combinations thereof.

A further aspect of the present invention relates to the peptides asdescribed above, preferably a peptide characterized by Formula IIa, morepreferably the peptide characterized by Formula IIb, i.e., iVR1-Cys, foruse as a medicament.

A further aspect of the present invention relates to the peptides asdescribed above, preferably a peptide characterized by Formula IIa, morepreferably the peptide characterized by Formula IIb, i.e., iVR1-Cys, orthe composition comprising said peptides as described above for use inthe treatment of a pathological condition associated with or caused byincorrect angiogenesis/neo-angiogenesis, i.e., a pathology in whichangiogenesis/neoangiogenesis is unregulated; it has preferably increasedand therefore needs to be inhibited.

Besides being useful in the treatment of said pathologies, the peptidesas described above, preferably a peptide characterized by Formula IIa,more preferably the peptide characterized by Formula IIb, i.e.,iVR1-Cys, or the composition comprising said peptides as described abovecan also be used for the follow-up of further alternative therapeutictreatments for said pathologies.

As already said previously, the angiogenesis/neoangiogenesis, as earlierdefined, is/are preferably dependent on/induced by/regulated by VEGFR1,or by the VEGFR1 pathway.

Said pathology/condition is preferably selected from:

-   -   neovascular eye diseases, preferably selected from: macular        edema, the wet form of age-related macular degeneration,        diabetic retinopathy, retinopathy of prematurity, retinopathy of        central retinal vein occlusion, vitreous hemorrhage and retinal        detachment and combinations thereof; and/or    -   solid or liquid tumors and/or tumor metastasis, said tumors        preferably being selected from: leukemias and lymphomas,        preferably acute lymphocytic leukemia, acute non-lymphocytic        leukemia, chronic lymphocytic leukemia, multiple myeloma,        Hodgkin's lymphoma, Hodgkin's disease, infantile or adult solid        tumors, brain tumors, neuroblastoma, retinoblastoma, Wilms        tumor, osteosarcomas and chondrosarcomas, lung tumors,        colorectal cancer, breast cancer, prostate cancer, uterine        cancer, ovarian cancer, urinary system cancer, bladder cancer,        tumor of the oral cavity, tumor of the pancreas, melanoma and        tumors of the skin, tumor of the stomach, tumor of the brain,        tumor of the thyroid, tumor of the larynx, tumor of the liver,        tumor of the testicles; and/or    -   diseases of the bones or joints, preferably selected from:        rheumatoid arthritis, synovitis, cartilage and/or bone        destruction, osteomyelitis, hypertrophy and/or hyperplasia of        the synovial tissue, formation of osteophytes, neoplasms and/or        metastases and combinations thereof; and/or    -   pathologies of blood vessels, preferably selected from:        atherosclerosis, hemangioma, hemangioendothelioma and        combinations thereof; and/or    -   skin diseases, preferably selected from: psoriasis, warts,        pyogenic granulomas, hair growth, Kaposi's sarcoma, keloids of        wounds, allergic edema, neoplasms and combinations thereof;        and/or    -   angiogenesis observed in pathologies of adipose tissue,        preferably obesity; and/or    -   diabetes and/or its consequences, preferably retinopathy and/or        diabetic foot; and/or    -   diseases of hematopoiesis, preferably AIDS and/or Kaposi's        sarcoma.

For the above-described medical purposes, the peptides and thecomposition of the invention can optionally be combined or also usedbefore or after already known drugs used to treat the above pathologies.

Furthermore, the peptides or the composition of the invention can beassociated with already known treatments of a surgical, radiotherapeuticor chemotherapeutic type which are used to treat the above pathologies.

The peptides of the present invention or the composition comprising saidpeptides as described above can be formulated so as to be administeredthrough any route. The route of administration is preferably selectedfrom: systemic route, preferably the oral route, gavage, sublingual orrectal route, the topical, subcutaneous, intramuscular, intravenous,intra-arterial, intraperitoneal, intradermal and intraepidermal route.

The peptides or the composition of the invention can be formulated as asolid, for example as pills, tablets, granules, soluble granules,pellets, beads, lozenges, and the like. Alternatively, the peptides orthe composition of the invention can be formulated as a liquid solution,for example to be administered by injection, inhalation or nebulization,or as drops or sprays.

The peptides of the present invention or the composition comprising saidpeptides as described above can be administered as a bolus.

The peptides of the present invention or the composition comprising saidpeptides as described above can be administered by means of medicaldevices, for example by means of stents, pump or patches.

The administration can preferably be continuous, by controlled releaseor by constant release, preferably using devices for ocular drugdelivery.

Administration by the oral route or gavage is particularly preferred. Infact, as previously described, the peptides of the present invention,iVR1 included, show to be effective in inhibitingangiogenesis/neoangiogenesis also when administered by gavage. They haveshown to be effective also for inhibiting angiogenesis/neoangiogenesisin the eye; in other words, when the peptides of the invention, iVR1included, were administered by gavage, an inhibition ofangiogenesis/neoangiogenesis in the eye was surprisingly observed. Theangiogenesis/neoangiogenesis being referred to is preferablyVEGFR1-dependent.

In the light of this scientific evidence, a further aspect of thepresent invention relates to the peptides of the invention, preferablyat least one peptide characterized by Figure IIa, more preferably thepeptide characterized by Formula IIb, i.e., iVR1-Cys, and/or the peptidecharacterized by Formula IIc, i.e., iVR1, or a composition comprisingsaid peptides administered orally or by gavage, for use in the treatmentof pathologies caused by or in any case associated with an incorrect,preferably increased, angiogenesis/neoangiogenesis, preferablyVEGFR1-dependent.

Said pathology/condition is preferably selected from:

-   -   neovascular eye diseases, preferably selected from: macular        edema, the wet form of age-related macular degeneration,        diabetic retinopathy, retinopathy of prematurity, retinopathy of        central retinal vein occlusion, vitreous hemorrhage and retinal        detachment and combinations thereof; and/or    -   solid tumors and/or tumor metastasis, said tumors preferably        being selected from: leukemias and lymphomas, preferably acute        lymphocytic leukemia, acute non-lymphocytic leukemia, chronic        lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma,        Hodgkin's disease, infantile or adult solid tumors, brain        tumors, neuroblastoma, retinoblastoma, Wilms tumor,        osteosarcomas and chondrosarcomas, lung tumors, colorectal        cancer, breast cancer, prostate cancer, uterine cancer, ovarian        cancer, urinary system cancer, bladder cancer, tumor of the oral        cavity, tumor of the pancreas, melanoma and tumors of the skin,        tumor of the stomach, tumor of the brain, tumor of the thyroid,        tumor of the larynx, tumor of the liver, tumor of the testicles;        and/or    -   diseases of the bones or joints, preferably selected from:        rheumatoid arthritis, synovitis, cartilage and/or bone        destruction, osteomyelitis, hypertrophy and/or hyperplasia of        the synovial tissue, formation of osteophytes, neoplasms and/or        metastases and combinations thereof; and/or    -   pathologies of blood vessels, preferably selected from:        atherosclerosis, hemangioma, hemangioendothelioma and        combinations thereof; and/or    -   skin diseases, preferably selected from: psoriasis, warts,        pyogenic granulomas, hair growth, Kaposi's sarcoma, keloids of        wounds, allergic edema, neoplasms and combinations thereof;        and/or    -   angiogenesis observed in pathologies of adipose tissue,        preferably obesity; and/or    -   diabetes and/or its consequences, preferably retinopathy and/or        diabetic foot; and/or    -   diseases of hematopoiesis, preferably AIDS and/or Kaposi's        sarcoma.

The peptide or the composition of the invention is administered to anyanimal that has need of it, preferably an animal in which there is aneed to inhibit VEGFR-1-dependent neoangiogenesis.

Said animal is preferably a mammal, more preferably it is a human being.

The effective dose of the peptide or of the composition as describedabove that is administered preferably ranges:

-   -   between 10 and 2000 mg/dose, preferably when administered        systemically, preferably by the systemic enteral route, more        preferably orally, sublingually or rectally; or    -   between 1 and 100 mg/dose when administered preferably        intravitreally.

Alternatively, the effective dose that is administered preferably rangesbetween 0.16 and 33.3 mg/kg of body weight.

The treatment program preferably provides for a single dose or multipledoses.

The sequences of the invention are annotated according to theinternational standard WIPO ST.25 and the description thereof wasdeveloped with the program Patent-In 3.5. A description of the sequencesis attached hereto.

In the present invention, the sequences identified in Table III and thesequences having an identity ranging from 80 to 99.9% are to beconsidered described.

TABLE III Sequence Name SEQ ID atcgaggtcc gcgggaggct cggagcgcgc Full-SEQ ID caggcggaca ctcctctcgg ctcctccccg length NO: 1gcagcggcgg cggctcggag cgggctccgg human ggctcgggtg cagcggccag cgggcgcctgVEGFR1 gcggcgagga ttacccgggg aagtggttgt mRNActcctggctg gagccgcgag acgggcgctc agggcgcggg gccggcggcg gcgaacgagaggacggactc tggcggccgg gtcgttggcc gcggggagcg cgggcaccgg gcgagcaggccgcgtcgcgc tcaccatggt cagctactgg gacaccgggg tcctgctgtg cgcgctgctcagctgtctgc ttctcacagg atctagttca ggttcaaaat taaaagatcc tgaactgagtttaaaaggca cccagcacat catgcaagca ggccagacac tgcatctcca atgcaggggggaagcagccc ataaatggtc tttgcctgaa atggtgagta aggaaagcga aaggctgagcataactaaat ctgcctgtgg aagaaatggc aaacaattct gcagtacttt aaccttgaacacagctcaag caaaccacac tggcttctac agctgcaaat atctagctgt acctacttcaaagaagaagg aaacagaatc tgcaatctat atatttatta gtgatacagg tagacctttcgtagagatgt acagtgaaat ccccgaaatt atacacatga ctgaaggaag ggagctcgtcattccctgcc gggttacgtc acctaacatc actgttactt taaaaaagtt tccacttgacactttgatcc ctgatggaaa acgcataatc tgggacagta gaaagggctt catcatatcaaatgcaacgt acaaagaaat agggcttctg acctgtgaag caacagtcaa tgggcatttgtataagacaa actatctcac acatcgacaa accaatacaa tcatagatgt ccaaataagcacaccacgcc cagtcaaatt acttagaggc catactcttg tcctcaattg tactgctaccactcccttga acacgagagt tcaaatgacc tggagttacc ctgatgaaaa aaataagagagcttccgtaa ggcgacgaat tgaccaaagc aattcccatg ccaacatatt ctacagtgttcttactattg acaaaatgca gaacaaagac aaaggacttt atacttgtcg tgtaaggagtggaccatcat tcaaatctgt taacacctca gtgcatatat atgataaagc attcatcactgtgaaacatc gaaaacagca ggtgcttgaa accgtagctg gcaagcggtc ttaccggctctctatgaaag tgaaggcatt tccctcgccg gaagttgtat ggttaaaaga tgggttacctgcgactgaga aatctgctcg ctatttgact cgtggctact cgttaattat caaggacgtaactgaagagg atgcagggaa ttatacaatc ttgctgagca taaaacagtc aaatgtgtttaaaaacctca ctgccactct aattgtcaat gtgaaacccc agatttacga aaaggccgtgtcatcgtttc cagacccggc tctctaccca ctgggcagca gacaaatcct gacttgtaccgcatatggta tccctcaacc tacaatcaag tggttctggc acccctgtaa ccataatcattccgaagcaa ggtgtgactt ttgttccaat aatgaagagt cctttatcct ggatgctgacagcaacatgg gaaacagaat tgagagcatc actcagcgca tggcaataat agaaggaaagaataagatgg ctagcacctt ggttgtggct gactctagaa tttctggaat ctacatttgcatagcttcca ataaagttgg gactgtggga agaaacataa gcttttatat cacagatgtgccaaatgggt ttcatgttaa cttggaaaaa atgccgacgg aaggagagga cctgaaactgtcttgcacag ttaacaagtt cttatacaga gacgttactt ggattttact gcggacagttaataacagaa caatgcacta cagtattagc aagcaaaaaa tggccatcac taaggagcactccatcactc ttaatcttac catcatgaat gtttccctgc aagattcagg cacctatgcctgcagagcca ggaatgtata cacaggggaa gaaatcctcc agaagaaaga aattacaatcagagatcagg aagcaccata cctcctgcga aacctcagtg atcacacagt ggccatcagcagttccacca ctttagactg tcatgctaat ggtgtccccg agcctcagat cacttggtttaaaaacaacc acaaaataca acaagagcct ggaattattt taggaccagg aagcagcacgctgtttattg aaagagtcac agaagaggat gaaggtgtct atcactgcaa agccaccaaccagaagggct ctgtggaaag ttcagcatac ctcactgttc aaggaacctc ggacaagtctaatctggagc tgatcactct aacatgcacc tgtgtggctg cgactctctt ctggctcctattaaccctct ttatccgaaa aatgaaaagg tcttcttctg aaataaagac tgactacctatcaattataa tggacccaga tgaagttcct ttggatgagc agtgtgagcg gctcccttatgatgccagca agtgggagtt tgcccgggag agacttaaac tgggcaaatc acttggaagaggggcttttg gaaaagtggt tcaagcatca gcatttggca ttaagaaatc acctacgtgccggactgtgg ctgtgaaaat gctgaaagag ggggccacgg ccagcgagta caaagctctgatgactgagc taaaaatctt gacccacatt ggccaccatc tgaacgtggt taacctgctgggagcctgca ccaagcaagg agggcctctg atggtgattg ttgaatactg caaatatggaaatctctcca actacctcaa gagcaaacgt gacttatttt ttctcaacaa ggatgcagcactacacatgg agcctaagaa agaaaaaatg gagccaggcc tggaacaagg caagaaaccaagactagata gcgtcaccag cagcgaaagc tttgcgagct ccggctttca ggaagataaaagtctgagtg atgttgagga agaggaggat tctgacggtt tctacaagga gcccatcactatggaagatc tgatttctta cagttttcaa gtggccagag gcatggagtt cctgtcttccagaaagtgca ttcatcggga cctggcagcg agaaacattc ttttatctga gaacaacgtggtgaagattt gtgattttgg ccttgcccgg gatatttata agaaccccga ttatgtgagaaaaggagata ctcgacttcc tctgaaatgg atggctcctg aatctatctt tgacaaaatctacagcacca agagcgacgt gtggtcttac ggagtattgc tgtgggaaat cttctccttaggtgggtctc catacccagg agtacaaatg gatgaggact tttgcagtcg cctgagggaaggcatgagga tgagagctcc tgagtactct actcctgaaa tctatcagat catgctggactgctggcaca gagacccaaa agaaaggcca agatttgcag aacttgtgga aaaactaggtgatttgcttc aagcaaatgt acaacaggat ggtaaagact acatcccaat caatgccatactgacaggaa atagtgggtt tacatactca actcctgcct tctctgagga cttcttcaaggaaagtattt cagctccgaa gtttaattca ggaagctctg atgatgtcag atacgtaaatgctttcaagt tcatgagcct ggaaagaatc aaaacctttg aagaactttt accgaatgccacctccatgt ttgatgacta ccagggcgac agcagcactc tgttggcctc tcccatgctgaagcgcttca cctggactga cagcaaaccc aaggcctcgc tcaagattga cttgagagtaaccagtaaaa gtaaggagtc ggggctgtct gatgtcagca ggcccagttt ctgccattccagctgtgggc acgtcagcga aggcaagcgc aggttcacct acgaccacgc tgagctggaaaggaaaatcg cgtgctgctc cccgccccca gactacaact cggtggtcct gtactccaccccacccatct agagtttgac acgaagcctt atttctagaa gcacatgtgt atttatacccccaggaaact agcttttgcc agtattatgc atatataagt ttacaccttt atctttccatgggagccagc tgctttttgt gattttttta atagtgcttt tttttttttg actaacaagaatgtaactcc agatagagaa atagtgacaa gtgaagaaca ctactgctaa atcctcatgttactcagtgt tagagaaatc cttcctaaac ccaatgactt ccctgctcca acccccgccacctcagggca cgcaggacca gtttgattga ggagctgcac tgatcaccca atgcatcacgtaccccactg ggccagccct gcagcccaaa acccagggca acaagcccgt tagccccagggatcactggc tggcctgagc aacatctcgg gagtcctcta gcaggcctaa gacatgtgaggaggaaaagg aaaaaaagca aaaagcaagg gagaaaagag aaaccgggag aaggcatgagaaagaatttg agacgcacca tgtgggcacg gagggggacg gggctcagca atgccatttcagtggcttcc cagctctgac ccttctacat ttgagggccc agccaggagc agatggacagcgatgagggg acattttctg gattctggga ggcaagaaaa ggacaaatat cttttttggaactaaagcaa attttagaac tttacctatg gaagtggttc tatgtccatt ctcattcgtggcatgttttg atttgtagca ctgagggtgg cactcaactc tgagcccata cttttggctcctctagtaag atgcactgaa aacttagcca gagttaggtt gtctccaggc catgatggccttacactgaa aatgtcacat tctattttgg gtattaatat atagtccaga cacttaactcaatttcttgg tattattctg ttttgcacag ttagttgtga aagaaagctg agaagaatgaaaatgcagtc ctgaggagag gagttttctc catatcaaaa cgagggctga tggaggaaaaaggtcaataa ggtcaaggga aaaccccgtc tctataccaa ccaaaccaat tcaccaacacagttgggacc caaaacacag gaagtcagtc acgtttcctt ttcatttaat ggggattccactatctcaca ctaatctgaa aggatgtgga agagcattag ctggcgcata ttaagcactttaagctcctt gagtaaaaag gtggtatgta atttatgcaa ggtatttctc cagttgggactcaggatatt agttaatgag ccatcactag aagaaaagcc cattttcaac tgctttgaaacttgcctggg gtctgagcat gatgggaata gggagacagg gtaggaaagg gcgcctactcttcagggtct aaagatcaag tgggccttgg atcgctaagc tggctctgtt tgatgctatttatgcaagtt agggtctatg tatttatgat gtctgcacct tctgcagcca gtcagaagctggagaggcaa cagtggattg ctgcttcttg gggagaagag tatgcttcct tttatccatgtaatttaact gtagaacctg agctctaagt aaccgaagaa tgtatgcctc tgttcttatgtgccacatcc ttgtttaaag gctctctgta tgaagagatg ggaccgtcat cagcacattccctagtgagc ctactggctc ctggcagcgg cttttgtgga agactcacta gccagaagagaggagtggga cagtcctctc caccaagatc taaatccaaa caaaagcagg ctagagccagaagagaggac aaatctttgt tcttcctctt ctttacatac gcaaaccacc tgtgacagctggcaatttta taaatcaggt aactggaagg aggttaaaca cagaaaaaag aagacctcagtcaattctct actttttttt ttttttccaa atcagataat agcccagcaa atagtgataacaaataaaac cttagctatt catgtcttga tttcaataat taattcttaa tcattaagagaccataataa atactccttt tcaagagaaa agcaaaacca ttagaattgt tactcagctccttcaaactc aggtttgtag catacatgag tccatccatc agtcaaagaa tggttccatctggagtctta atgtagaaag aaaaatggag acttgtaata atgagctagt tacaaagtgcttgttcatta aaatagcact gaaaattgaa acatgaatta actgataata ttccaatcatttgccattta tgacaaaaat ggttggcact aacaaagaac gagcacttcc tttcagagtttctgagataa tgtacgtgga acagtctggg tggaatgggg ctgaaaccat gtgcaagtctgtgtcttgtc agtccaagaa gtgacaccga gatgttaatt ttagggaccc gtgccttgtttcctagccca caagaatgca aacatcaaac agatactcgc tagcctcatt taaattgattaaaggaggag tgcatctttg gccgacagtg gtgtaactgt atgtgtgtgt gtgtgtgtgtgtgtgtgtgt gtgtgtgggt gtatgtgtgt tttgtgcata actatttaag gaaactggaattttaaagtt acttttatac aaaccaagaa tatatgctac agatataaga cagacatggtttggtcctat atttctagtc atgatgaatg tattttgtat accatcttca tataataaacttccaaaaac aca mvsywdtgvl lcallsclll tgsssgsklk Full- SEQ IDdpelslkgtq himqagqtlh lqcrgeaahk length NO: 2wslpemvske serlsitksa cgrngkqfcs human tltlntaqan htgfysckyl avptskkketVEGFR1 esaiyifisd tgrpfvemys eipeiihmte proteingrelvipcrv tspnitvtlk kfpldtlipd gkriiwdsrk gfiisnatyk eiglltceatvnghlyktny lthrqtntii dvqistprpv kllrghtlvl nctattplnt rvqmtwsypdeknkrasvrr ridqsnshan ifysvltidk mqnkdkglyt crvrsgpsfk svntsvhiydkafitvkhrk qqvletvagk rsyrlsmkvk afpspevvwl kdglpateks aryltrgysliikdvteeda gnytillsik qsnvfknlta tlivnvkpqi yekavssfpd palyplgsrqiltctaygip qptikwfwhp cnhnhsearc dfcsnneesf ildadsnmgn riesitqrmaiiegknkmas tlvvadsris giyiciasnk vgtvgrnisf yitdvpngfh vnlekmptegedlklsctvn kflyrdvtwi llrtvnnrtm hysiskqkma itkehsitln ltimnvslqdsgtyacrarn vytgeeilqk keitirdqea pyllrnlsdh tvaisssttl dchangvpepqitwfknnhk iqqepgiilg pgsstlfier vteedegvyh ckatnqkgsv essayltvqgtsdksnleli tltctcvaat lfwllltlfi rkmkrsssei ktdylsiimd pdevpldeqcerlpydaskw efarerlklg kslgrgafgk vvqasafgik ksptcrtvav kmlkegataseykalmtelk ilthighhln vvnllgactk qggplmvive yckygnlsny lkskrdlfflnkdaalhmep kkekmepgle qgkkprldsv tssesfassg fqedkslsdv eeeedsdgfykepitmedli sysfqvargm eflssrkcih rdlaarnill sennVVkicd fglardiyknpdyvrkgdtr lplkwmapes ifdkiystks dvwsygvllw eifslggspy pgvqmdedfcsrlregmrmr apeystpeiy qimldcwhrd pkerprfael veklgdllqa nvqqdgkdyipinailtgns gftystpafs edffkesisa pkfnsgssdd vryvnafkfm sleriktfeellpnatsmfd dyqgdsstll aspmlkrftw tdskpkaslk idlrvtsksk esglsdvsrpsfchsscghv segkrrftyd haelerkiac cspppdynsv vlystppiatggtcagct actgggacac cggggtcctg Soluble SEQ IDctgtgcgcgc tgctcagctg tctgcttctc human NO: 3acaggatcta gttcaggttc aaaattaaaa VEGFR1 gatcctgaac tgagtttaaa aggcacccagmRNA cacatcatgc aagcaggcca gacactgcat ctccaatgca ggggggaagc agcccataaatggtctttgc ctgaaatggt gagtaaggaa agcgaaaggc tgagcataac taaatctgcctgtggaagaa atggcaaaca attctgcagt actttaacct tgaacacagc tcaagcaaaccacactggct tctacagctg caaatatcta gctgtaccta cttcaaagaa gaaggaaacagaatctgcaa tctatatatt tattagtgat acaggtagac ctttcgtaga gatgtacagtgaaatccccg aaattataca catgactgaa ggaagggagc tcgtcattcc ctgccgggttacgtcaccta acatcactgt tactttaaaa aagtttccac ttgacacttt gatccctgatggaaaacgca taatctggga cagtagaaag ggcttcatca tatcaaatgc aacgtacaaagaaatagggc ttctgacctg tgaagcaaca gtcaatgggc atttgtataa gacaaactatctcacacatc gacaaaccaa tacaatcata gatgtccaaa taagcacacc acgcccagtcaaattactta qaggccatac tcttgtcctc aattgtactg ctaccactcc cttgaacacgagagttcaaa tgacctggag ttaccctgat gaaaaaaata agagagcttc cgtaaggcgacgaattgacc aaagcaattc ccatgccaac atattctaca gtgttcttac tattgacaaaatgcagaaca aagacaaagg actttatact tgtcgtgtaa ggagtggacc atcattcaaatctgttaaca cctcagtgca tatatatgat aaagcattca tcactgtgaa acatcgaaaacagcaggtgc ttgaaaccgt agctggcaag cggtcttacc ggctctctat gaaagtgaaggcatttccct cgccggaagt tgtatggtta aaagatgggt tacctgcgac tgagaaatctgctcgctatt tgactcgtgg ctactcgtta attatcaagg acgtaactga agaggatgcagggaattata caatcttgct gagcataaaa cagtcaaatg tgtttaaaaa cctcactgccactctaattg tcaatgtgaa accccagatt tacgaaaagg ccgtgtcatc gtttccagacccggctctct acccactggg cagcagacaa atcctgactt gtaccgcata tggtatccctcaacctacaa tcaagtggtt ctggcacccc tgtaaccata atcattccga agcaaggtgtgacttttgtt ccaataatga agagtccttt atcctggatg ctgacagcaa catgggaaacagaattgaga gcatcactca gcgcatggca ataatagaag gaaagaataa gcttccaccagctaacagtt ctttcatgtt gccacctaca agcttctctt ccaactactt ccatttcctt ccgtgamvsywdtgvl lcallsclll tgsssgsklk Soluble SEQ IDdpelslkgtq himqagqtlh lqcrgeaahk human NO: 4wslpemvske serlsitksa cgrngkqfcs VEGFR1 tltlntaqan htgfysckyl avptskkketprotein esaiyifisd tgrpfvemys eipeiihmtegrelvipcrv tspnitvtlk kfpldtlipd gkriiwdsrk gfiisnatyk eiglltceatvnghlyktny ithrqtntii dvqistprpv kllrghtlvl nctattplnt rvqmtwsypdeknkrasvrr ridqsnshan ifysvltidk mqnkdkglyt crvrsgpsfk svntsvhiydkafitvkhrk qqvletvagk rsyrlsmkvk afpspevvwl kdglpateks aryltrgysliikdvteeda gnytillsik qsnvfknlta tlivnvkpqi yekavssfpd palyplgsrqiltctaygip qptikwfwhp cnhnhsearc dfcsnneesf ildadsnmgn riesitqrmaiiegknklpp anssfmlppt sfssnyfhflp

EXAMPLE Dose-Dependent Inhibition of VEGF-A/VEGFR1 and PIGF/VEGFR-1Interaction.

The assay to test the binding of PIGF or VEGF-A with the VEGFR-1receptor is based on the ELISA method [Ponticelli et al., JBC. 2008 Dec.5; 283(49):34250-9] and was performed using reagents acquired from R&DSystems.

The human recombinant receptor VEGFR-1, in particular the formconsisting of the seven extracellular domains of the receptor fused tothe Fc domain of human IgG (R&D Systems, cat No. 321-FL), was made toadhere in the wells of 96-well microplates at a concentration of 0.5μg/ml in PBS pH 7.5 (100 μl/well) for 16 hours at room temperature (RT).After the non-specific binding sites had been blocked in the wells usinga buffer solution consisting of PBS pH 7.5 containing 3% BSA, 5 ng/ml ofrecombinant PIGF (R&D Systems, cat No. 264-PG), or 5 ng/ml ofrecombinant VEGF-A (R&D Systems, cat No. 293-VE) of human origin in PBET(PBS pH 7.5, BSA 0.1%, EDTA 5 mM, Tween 0.004%) were added to the wellswith the adhered receptor.

Simultaneously with the ligands, i.e., PIGF or VEGF-A, graduated dosesof iVR1, iVR1-Cys or a control peptide (PC-[(S-Ser)-(S-Ala)-(S-Cha)tripeptide with a tetrameric structure identical to the structure of theiVR1 peptides]) were added at concentrations comprised between 780 and50000 nM. The binding reaction was conducted for one hour at 37° C.,followed by one hour at room temperature.

At the end of the binding and/or competition step, anti-human-PIGFbiotinylated polyclonal antibodies (R&D Systems, cat No. BAF264) oranti-human-VEGF-A (R&D Systems, cat No. BAF293) were added to the wellsat the concentration of 300 ng/ml in PBET. After one hour of incubationat 37° C. followed by one hour at room temperature, an HRP-conjugatedavidin-streptavidin system (Vectastain elite ABC kit) and a substratefor HRP (o-phenylenediamine—Sigma, cat No. P1526) were added to thewells. Quantization was performed by determining the absorbance at 490nM.

Any inhibitory activity of the peptides was expressed in terms of % ofresidual binding, comparing the data obtained for the binding of PIGF orVEGF-A to the receptors in the presence of the tetrameric peptides withthose in the absence of the same. iVR1 represented the positive controlof the inhibition of the PIGF/VEGFR-1 or VEGF-A/VEGFR-1 interaction.

The results are given in Tables IV and V and show that iVR1-Cysdemonstrated a capacity to inhibit the interaction both of PIGF andVEGF-A with VEGFR-1 in a dose-dependent manner.

The concentration at which iVR1-Cys is capable of inhibiting theinteraction of PIGF with VEGFR-1 by 50% (IC₅₀) is below 1000 nM, whereasthe IC50 for VEGF-A/VEGFR-1 is close to or just above 1000 nM.Therefore, iVR1-Cys has an inhibitory capacity that is about 10 timesgreater than that of iVR1, and it is thus expected that it can be usedat doses that are 10 times smaller in the same in vitro and in vivoexperimental protocols relating to angiogenesis/neoangiogenesisinhibition in order to obtain the same effects as obtained with iVR1.

PC gives no inhibition.

TABLE IV Dose-dependent inhibition of PIGF/VEGFR-1 interaction binding %peptides [nM] iVR1-Cys iVR1 PC 780 83.82 99.36 98.88 1560 35.50 101.32102.35 3125 25.54 96.10 99.53 6250 14.09 68.49 93.82 12500 12.59 44.5787.21 25000 10.27 36.02 88.55 50000 9.18 26.26 90.16

TABLE V Dose-dependent inhibition of VEGF-A/VEGFR-1 interaction binding% peptides [nM] iVR1-Cys iVR1 PC 780 92.32 98.36 100.09 1560 43.71 96.21102.59 3125 21.70 93.93 95.20 6250 12.50 66.00 91.37 12500 9.81 37.02100.04 25000 7.99 21.45 93.44 50000 6.50 8.60 87.93

The capacity of tetrameric peptides having formula (II), but with Y2different from D-cysteine, to inhibit VEGF-A/VEGFR-1 binding wasassessed with the binding assay described above. Y2 of the peptides andthe respective IC₅₀ of inhibition of VEGF-A/VEGFR-1 interaction areindicated in Table VI.

TABLE VI IC₅₀ of inhibition of the interaction VEGF-A/VEGFR-1 Y2 IC₅₀,μM L-cysteine 2.03 ± 0.2 L-methionine 2.15 ± 1.0 D-methionine 2.89 ± 1.2L-methionine sulfoxide 15.7 ± 2.1 L-methionine sulfone 4.62 ± 1.4D-serine 2.43 ± 03  L-serine 2.90 ± 0.5 D-threonine 2.60 ± 0.6L-threonine 1.29 ± 0.5

Inhibition of PIGF-Induced Phosphorylation of VEGFR-1.

An assay of the PIGF-induced phosphorylation of the receptor VEGFR-1 wasperformed in order to evaluate the inhibitory capacity of the peptideiVR1-Cys and compare its activity to that of iVR1.

For the activation of VEGFR-1, use was made of a cell lineover-expressing the receptor, called 293-VEGFR-1, obtained by stabletransfection from HEK-293 cells (Errico, M. et al. 2004 JBC, 279:43929-43939).

For this purpose, the 293-VEGFR-1 cells were cultured until reachingsubconfluence and the cells were subsequently ‘starved’, bykeeping/incubating them in the culture medium without serum for at least16 hours.

At the end of the starvation step, the culture medium was removed andthe cell monolayers were incubated with Na₃VO₄ 100 μM for 5 minutes inorder to inhibit the activity of the endogenous phosphatase.

The cells were then stimulated with PIGF (1) alone at 20 ng/ml in themedium used for the starvation for 10 minutes at 37° C. and (2) in thepresence of the peptides at the concentration of 5 μM.

An anti-human-PIGF neutralizing monoclonal antibody (Thrombogenics) wasused at a concentration of 3.3 nM as an inhibition control. PBS was usedas a negative control.

At the end of incubation, the cells were washed with cold Na₃VO₄ 100 μMand then lysed in the buffer composed of Tris-HCl 20 mM pH 8, EDTA 5 mM,NaCl 150 mM, 1% Triton-X100, 10% glycerol, zinc acetate 10 mM, Na₃VO₄100 μM and a mixture of protease inhibitors and incubated for 1 hour at4° C. under gentle stirring. At the end, the cell lysates werecentrifuged at 12000×g for 15 minutes to remove the cellular debris.Quantization of the extracts was performed with the Bradford methodusing a Bio-Rad reagent. 100 μg of every protein extract were loaded onSDS-PAGE reducing to 8.5%, and then the standard method for analyzingproteins was carried out by western blotting.

The anti-p-VEGFR-1 antibody (R&D Systems, cat. No. AF4170), diluted1:500, was used to detect the phosphorylated VEGFR-1, whilstnormalization was carried out by detecting the non-phosphorylated formof the receptor using the anti-VEGFR-1 antibody (Sigma-Aldrich, cat. No.V4262) diluted 1:500.

As shown in FIG. 1, the peptide iVR1-Cys, used at a concentration about5 times higher (5000 nM) than its IC₅₀ determined in the binding assays(see Example 1), brings about a powerful inhibition of phosphorylationof the receptor, similar to the one obtained with the neutralizingantibody and decidedly greater than the one obtained with iVR1 using thesame concentration.

Inhibition of Choroidal Neovascularization by Intravitreal and Oral(Gavage) Administration of iVR1 and iVR1-Cys.

The experimental model of laser-induced choroidal neovascularizationentails generating damage to Bruch's membrane, which separates thechoroid from the pigmented epithelium of the retina (RPE). The damage isprovoked by laser-induced burning, which causes the perforation ofBruch's membrane, thus activating chorioretinal vascularization, thegrowth of new vessels which, starting from the choroid, invade theoverlying retinal tissue. This mouse model sums up the maincharacteristics of the exudative form of human age-related maculardegeneration (AMD) and is in fact commonly used as a preclinical modelof AMD. It enables an assessment of the anti-angiogenic activity of themolecules of interest.

In order to be able to visualize the ocular fundus of the mouse andinduce damage with the laser, the Micron IV integrated system was used,following the experimental procedure described below.

First of all, dilatation of the animal's pupil was induced by applying0.5% Tropicamide eye drops. The animal was then anaesthetized byintraperitoneal injection of a solution of ketamine and xylazine (80mg/Kg and 10 mg/Kg, respectively). Once sedated, the animal was placedon the stand and a hydroxypropyl methylcellulose 2.5% aqueous solutionwas applied on both eyes. It has the dual function of preventingdehydration of the cornea and improving visualization of the ocularfundus by placing the camera lens of the Micron IV in contact with thesolution (a procedure similar to the one used in microscopy withimmersion objectives).

In order to induce damage with the laser, first of all the laser pointeris activated and focused so as to apply the laser beam using the RPElayer as a reference. The area where the laser beam is applied must bedistant from the main vessels of the retina in order to prevent possiblehemorrhaging. The efficiency of the burning at the level of Bruch'smembrane is confirmed by the formation of a bubble immediately afterapplication of the laser beam. The conditions of application of thelaser beam were 200 mW of power for 100 msec.

From data present in the literature, well summarized in the article byLambert et al. (Nature Protocols, 2013, 8:2197), it is known that themaximum neo-vascularization in this experimental model is obtained sevendays after the damage.

C57B16/J mice were used, n=5 per group. At the end of the procedure ofinducing damage with the laser, an intravitreal injection wasimmediately performed and 10 and 50 μg of iVR1-Cys or iVR1, and 50 μg ofPC in 1 μL of DMSO were administered using a Hamilton syringe with a 32g needle. As a control DMSO was injected on its own.

After seven days the animals were sacrificed and the eyes wereenucleated and fixed in 4% paraformaldehyde. Subsequently, the frontsegment of the eye, consisting of the: cornea, iris and crystalline wasremoved under a stereo microscope. The remaining part, defined‘eye-cups’ or posterior segment consisting of: sclera, choroid, RPE andretina was incubated in the presence of 0.7% FITC—Griffoniasimplicifolia Isolectin B4 (Vector Laboratories, Burlingame, Calif.) forsixteen hours. After a series of washes, the retina is removed and fourcuts are made on the RPE/choroid, which enables mounting on the slidefor observation under a fluorescence microscope. Quantization ofchoroidal neovascularization is performed in terms of volume. In orderto assess the volume of every spot, a series of images is acquired(Z-Satcks, about 20-25 image), each with a thickness of 1 μm, from theupper surface to the deepest focal plane, at the level of the RPE cells.The volume of fluorescence is measured by means of the ImageJ program(NIH, Bethesda, Md.), taking the sum of the areas of fluorescence ofevery single plane.

Quantization of CNV was performed on n=12 and 15 spots for iVR1 10 μgand 50 μg; n=10 and 8 spots for iVR1-Cys 10 μg and 50 μg; n=15 spots forPC and n=14 spots for DMSO. The results given in FIG. 2 show that bothpeptides are capable of bringing about a dose-dependent inhibition ofneovascularization. With the higher dose (50 μg), a powerful,significant and comparable neovascularization inhibition capacity wasobtained: iVR1-Cys −75.9% and −74.6% vs the vehicle and PC (p>0.002);iVR1 −73.9% and −76.5% vs the vehicle and PC (p>0.0002).

At the dosage of 10 μg, iVR1 brings about a 37.8% and 39.3% inhibitionof neovascularization vs the vehicle and PC (p<0.05), whereas iVR1-Cysbrings about a 48.9% and 51.0% inhibition vs the vehicle and PC(p<0.02). At a low concentration, therefore, the peptide iVR1-Cysdemonstrates a greater inhibition effectiveness than the peptide iVR1,as it brings about a further 19.3% reduction of neovascularization. Itis thus possible that the maximum threshold of the inhibitory capacityof the peptides was reached at the higher dosage used.

For the experiments on oral administration (gavage), choroidalneovascularization was induced in C57Bl6/J mice, n=5 animals per group,following the experimental procedure previously described. Theadministration of the peptides iVR1 and iVR1-Cys and of the vehiclebegan immediately after induction of the damage, as soon as the animalsrecovered from the anesthesia, twice a day for the seven days providedfor by the experimental protocol. The peptides were administered at 50mg/Kg, on the basis of the data obtained previously for the peptide iVR1administered intraperitoneally (Cicatiello et al. 2015, Oncotarget, 6,10563-10576).

To enable oral administration to be performed, the peptides weredissolved in DMSO, and then mixed with Nutilis food thickener, so as tohave a final mixture consisting of 9 parts Nutilis and 1 part DMSO.

The substances were prepared at a concentration such as to make itpossible to use, for every single administration, 200 μl of the 9:1Nutilis/substance in DMSO mixture, which was administered directly intothe animal's stomach using a suitable needle for gavage with a 20 gaugeopening. In the control group, 200 μI of the 9:1 Nutilis/DMSO mixturewere administered.

At the end of the experiment, the animals were sacrificed, the eyes wereremoved and dissected to isolate the RPE-choroid and to determine thevolume of CNV by immunofluorescence analysis, as described below.

Quantization of CNV was performed on n=18 spots for iVR1-Cys, n=20 spotsfor iVR1 and n=10 spots for the vehicle.

The results are given in FIG. 3 and demonstrate that the peptideiVR1-Cys is capable of inducing a significant inhibition ofneovascularization compared to the vehicle (−45.9%, p=0.007), at levelssimilar to those observed for iVR1 (−49.7%, p=0.001).

Serum Protease Stability of iVR1-Cys.

The stability of the peptide iVR1-Cys in 10% serum (fetal calf serum,FCS) in a 50 mM phosphate buffer solution, pH 7.3, at 168 h wasdetermined as described by Ponticelli et al., relying on a method basedon RP-HPLC chromatography, described therein [Ponticelli et al., J BiolChem. 2008 Dec. 5; 283(49):34250-9].

The reference curve was constructed by dissolving the compound in DMSOat increasing concentrations of between 0.1 μmol/L and 1000 μmol/L inorder to have complete dissolution. The concentration of the moleculeleft in contact with 10% FCS at the initial concentration of 10 μmol/Lwas then determined by drawing 3 aliquots at time t=0, then every hourin the first 12 hours and then at 24, 72, 120, and 168 h. The aliquotswere diluted 1:1 with acetic acid 0.1 M in order to detach any peptidebound to the albumin, centrifuged to remove any precipitated materialsand analyzed by RP-HPLC under the conditions reported in Ponticelli etal. The amount of residual peptide detected in the aliquots, expressedas a percentage relative to the initial amount, was plotted as afunction of time. The results are shown in Table VII as the mean of thethree determinations±the standard deviation (SD).

TABLE VII % residual Time (h) peptide ± SD  0 101 ± 4   1 100 ± 11   299 ± 7   3 98 ± 5   4 97 ± 4   5 96 ± 5   6 99 ± 6   7 95 ± 8   8 96 ±5   9 95 ± 6   10 94 ± 10  11 94 ± 8   12 93 ± 4   24 93 ± 6   72 92 ±7  120 91 ± 5  168 92 ± 8 

1. An isolated peptide characterized by the following formula (II){{{[Y1-Glu-Cys(Bzl)-Cha]2-Z1}i-Z2}j-Z3}z-Y2-Y3   (Formula II) wherein Y1is the amino-terminal peptide function (NH₂) or at least one chemicalgroup selected from: D-Alanine, D-Aspartic Acid, D-Valine, D-GlutamicAcid, L-Cyclohexyl-alanine, D-Phenylalanine, D-Threonine, D-Methionine,D-Lysine, D-Cysteine(S-acetamidomethyl), D-Tyrosine, D-Proline,D-Leucine, D-Arginine, D-Asparagine, D-Isoleucine,D-Arginine(N^(r)-Tosyl), D-Serine, L-Cysteine(S-benzyl),L-Cysteine(S-acetamidomethyl), D-Histidine, D-Glutamine, D-Tryptophan,L-Glutamic-(β-allyl) Acid, β-Alanine, L-Cysteine(S-p-methyl-benzyl),L-Cysteine(S-tert-butyl), L-Methionine-sulfone, L-Methionine-sulfoxide,Glycine, and combinations thereof; Glu indicates glutamic acid; Cys(Bzl)indicates benzyl cysteine; Cha indicates cyclohexylalanine; Y2 isselected from: an α-amino acid characterized by at least one thiol orthioether group; and R-Glu-S-Cys(Bzl)-S-Cha tripeptide; Y3 is selectedfrom: a carboxylic group, a carboxyamide group, an N-methyl-substitutedcarboxyamide or di-substituted N, N-dimethyl group, a hydroxyl group anda hydrogen; Z1, Z2 and Z3 indicate a trifunctional molecule, i is 4, 2or 1; j is 2, 1 or 0; and z is 1 or 0, with the condition that when i=4,j=2 and z=1; when i=2, j=1 and z=0; when i=1, j=z=0; when j=0 the Z2group is omitted and when z=0 the Z3 group is omitted.
 2. The peptideaccording to claim 1, wherein said trifunctional molecule ischaracterized by the following formula (III):

wherein k is an integer between 1 and 4, B is an amino group, or ahydroxyl group.
 3. The peptide according to claim 1, wherein at leasttwo of Z1, Z2, and Z3 are joined together by means of amide bonds or anester bond in order to form a branched structure.
 4. The peptideaccording to claim 1 characterized by formula (IIb), wherein:

Y1 is a hydrogen atom; Y2 is a D-cysteine; Y3 is an unsubstitutedprimary amide group Z1, Z2 and Z3 being as defined above; i is equal to2; j is equal to 1; and z is equal to zero or absent.
 5. A compositioncomprising at least one peptide according to claim 1 and at least onefurther pharmaceutically accepted ingredient.
 6. (canceled)
 7. A methodof treating a pathological condition associated with or caused by anunregulated angiogenesis/neo-angiogenesis, comprising administering thepeptide of claim 1 to a mammal in need thereof.
 8. The method accordingto claim 7, wherein said pathological condition is selected from:neovascular eye diseases; solid tumors, tumor metastasis, or acombination thereof; diseases of the bones or joints; skin diseases;angiogenesis observed in pathologies of adipose tissue; diabetes or itsconsequences; diseases of hematopoiesis; or combinations thereof. 9.(canceled)
 10. The peptide of claim 1, wherein: Glu indicates glutamicacid in an absolute configuration R on the Cα of the amino acid (R-Glu);Cys(Bzl) indicates benzyl cysteine in an absolute configuration S on theCα of the amino acid containing a sulfur-linked benzyl group of theamino acid (S-benzyl-cysteine/S-Cys (Bzl) side chain; Cha indicatescyclohexylalanine in an absolute configuration S on the Cα of the aminoacid (5-cyclohexylalanine/S-Cha); and Y2 is D-cysteine, L-cysteine,L-homocysteine, D-homocysteine, D-cysteine-S-methyl,L-cysteine-S-methyl, D-cysteine-S-ethyl, L-cysteine-S-ethyl,L-methionine, D-methionine, D-cysteine-S-benzyl, L-cysteine-S-benzyl,L-methionine sulfoxide, D-methionine sulfoxide, L-methionine sulfone,D-methionine sulfone, D-serine, L-serine, D-serine-O-methyl,L-serine-O-methyl, D-serine-O-ethyl, L-serine-O-ethyl,D-serine-O-benzyl, L-serine-O-benzyl, D-threonine, L-threonine,D-threonine-O-methyl, L-threonine-O-methyl, D-threonine-O-ethyl,L-threonine-O-ethyl, D-threonine-O-benzyl, L-threonine-O-benzyl, or acombination thereof.
 11. The peptide of claim 2, wherein B is anS-lysine or an R-lysine.
 12. The method of claim 7, wherein theangiogenesis/neo-angiogenesis is VEGFR1-dependent.
 13. The method ofclaim 8, wherein the pathological condition is: macular edema, the wetform of age-related macular degeneration, diabetic retinopathy,retinopathy of prematurity, central retinal vein occlusion, vitreoushemorrhage, retinal detachment, or a combination thereof.
 14. The methodof claim 8, wherein the pathological condition is: a leukemia, alymphoma, acute lymphocytic leukemia, acute non-lymphocytic leukemia,chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma,Hodgkin's disease, infantile or adult solid tumors, brain tumors,neuroblastoma, retinoblastoma, Wilms tumor, osteosarcomas andchondrosarcomas, lung tumors, colorectal cancer, breast cancer, prostatecancer, uterine cancer, ovarian cancer, urinary system cancer, bladdercancer, tumor of the oral cavity, tumor of the pancreas, melanoma andtumors of the skin, tumor of the stomach, tumor of the brain, tumor ofthe thyroid, tumor of the larynx, tumor of the liver, tumor of thetesticles, or a combination thereof.
 15. The method of claim 8, whereinthe pathological condition is: rheumatoid arthritis, synovitis,cartilage destruction, bone destruction, osteomyelitis, hypertrophyand/or hyperplasia of the synovial tissue, formation of osteophytes,neoplasms, metastases, or a combination thereof.
 16. The method of claim8, wherein the pathological condition is: atherosclerosis, hemangioma,hemangioendothelioma, or a combination thereof.
 17. The method of claim8, wherein the pathological condition is: psoriasis, warts, pyogenicgranulomas, hair growth, Kaposi's sarcoma, keloids of wounds, allergicedema, neoplasms, or a combination thereof.
 18. The method of claim 8,wherein the pathological condition is: obesity, diabetic retinopathy,diabetic foot, or a combination thereof.
 19. The method of claim 8,wherein the pathological condition is: AIDS, Kaposi's sarcoma, or acombination thereof.
 20. The method of claim 12, wherein VEGFR1 isactivated.